PROJECT SUMMARY/ABSTRACT Structural biology of RNA splicing. RNA splicing is a fundamental biological mechanism essential for the survival of every eukaryotic organism. It involves the excision of non-coding introns from pre-messenger RNAs and ligation of the adjacent exons to generate mature mRNA for translation by the ribosome. Spliceosomal introns comprise ~30% of the human genome and there is relatively little known about the precise structural details of their mechanism of excision catalyzed by the spliceosome. Group II introns are considered to be the ancestors of spliceosomal introns, therefore we are using the group II intron as a tractable model system to gain detailed molecular insight into eukaryotic splicing. Previously, the PI worked to determine the first crystal structure of a group II intron. This structure revealed that introns splice via a two-metal ion mechanism. However, this structure was of a hydrolytic, linear group II intron that was missing an important region called domain 6. This crucial domain contains the bulged adenosine responsible for forming the branched RNA known as a lariat. The lariat consists of an unusual 2'-5' phosphodiester bond between the ribose sugar of the bulged adenosine and the 5' end of the intron. The analogous motif in spliceosomal introns is called the branch site. Mutations in the branch site lead to defects in lariat formation that are the cause of several human diseases. Lariat formation is essential for proper splice site selection by the spliceosome and is considered to be a characteristic hallmark of eukaryotic splicing. Our long-term goal is to gain knowledge of eukaryotic splicing mechanisms by studying a lariat-forming group II intron. We will use biochemical and structural approaches to: 1) Determine the structural basis for lariat formation. 2) Analyze catalytic intermediates in the splicing pathway to determine the conformational changes that are associated with the progression of splicing. This is expected to have direct parallels with RNA splicing catalyzed by the spliceosome in eukaryotes. PUBLIC HEALTH RELEVANCE: NARRATIVE The goal of this project is to gain detailed structural insight into eukaryotic RNA splicing. Defects in RNA splicing account for ~15% of all human diseases. We aim to use the molecular knowledge of the splicing reaction as a foundation for the development of therapeutic treatments to treat these conditions.